The present invention relates to the proteolytic digestion of whole antibodies to obtain antibody fragments therefrom. In particular, the present invention relates to the preparation of F(ab').sub.2 antibody fragments derived from papain digested whole IgG antibodies.
The use of monovalent [Fab and Fab'] and divalent [F(ab').sub.2 ] antibody fragments derived from whole IgG antibodies is well established. For example, such fragments have been employed as labeled immunoassay reagents [Ullman, et al, Methods in Enzymology, Vol. 74, p 28 (1981); Inoue, et al., Analytical Letters, vol. 18, p. 1331 (1985); and German Patent Application No. DE 3430905] and as immunotherapeutic agents [Smith, et al., Antibodies in Human Diagnosis and Therapy, Haber and Krause (Eds.) , Raven Press, New York, N.Y., p. 365 (1977)]. In the area of immunodiagnostics, such monovalent antibody fragments have been shown to have certain advantages over reagents employing intact or whole IgG antibodies including, for example, diminished interference with samples containing rheumatoid factor and/or anti-species IgG antibodies. Independent of the ultimate use, preservation of the molecular integrity of antibodies and their binding activity during reagent synthesis and purification is crucial because the degradation thereof can result in altered antibody performance, and immunoassay performance would suffer as well. Where such intact or whole IgG antibodies are employed as an in vivo immunotherapeutic, serum sickness could occur.
The proteolytic digestion of whole or intact IgG antibodies to obtain F(ab').sub.2 antibody fragments therefrom is well known according to methods known in the art. The proteolytic enzyme pepsin is commonly used for this purpose. For example, F(ab').sub.2 antibody fragments can be prepared by pepsin digestion of whole mouse IgG antibodies [Lamoyi, et al, J. Immunol. Methods, Vol. 56, p. 235 (1983)] or rat IgG antibodies [Rousseaux, et al., J. Immunol. Methods, Vol. 64, p. 141 (1983)]. However, some antibodies, particularly monoclonal antibodies, are extremely sensitive to pepsin digestion and, in such cases, are rapidly degraded to antibody fragments having reduced or no binding activity. In order to avoid such degradation IgG molecules can instead be digested with preactivated papain [Parham, et al., J. Immunol. Methods, Vol. 53, p. 133, (1982)] to yield F(ab').sub.2 and F.sub.c fragments.
Although papain can be employed to digest pepsin-sensitive antibodies as described above, it has nevertheless been observed that even papain digestion can result in a significant increase in the degradation of the molecular integrity of antibody fragments derived therefrom. For example, such degradation results in a decreased number of available sulfhydryl groups in the hinge region and, in some instances, no sulfhydryl groups. Therefore, in such instances where it is desirable to digest whole IgG molecules with papain as described above, the prior art methods do not enable the preparation of intact active F(ab').sub.2 antibody fragments from those whole IgG antibodies which are otherwise susceptible to degradation and inactivation by papain digestion.
Accordingly, it is an object of the present invention to provide a papain-free F(ab').sub.2 antibody fragment preparation derived from papain digested whole IgG antibody.
Another object of the present invention is to provide an F(ab').sub.2 antibody fragment preparation from which Fab' antibody fragments can be obtained having available free sulfhydryl groups in the hinge region.